Cholera toxin (CT) is composed of two subunits: a toxigenic A subunit (CTA), and a binding B subunit (CTB). The latter mediates binding of CTA to the surface of eukaryotic cells via its interaction with the monosialoganglioside GM1. The type I heat-labile enterotoxin of Escherichia coli (LT) is closely related to CT. It has an identical subunit structure and function, and shares 80% amino acid homology with CT. Although LT is very similar to CT in structure and function, it binds to a broader array of ligands than CT, including glycolipids and glycoproteins.
The 3-D crystal structure has been determined for both CT and LT. In addition, the crystal structure of CTB complexed with the GM1-pentasaccharide has also been determined. The crystal structure of this complex has revealed all interactions between the GM1 head group and the receptor binding site of CTB without any indication of the presence of a divalent ion binding site. Also, no biochemical evidence indicating that cations mediate ganglioside binding has previously been reported. However, some of the receptors for these enterotoxins have only been recently identified. Thus, it is possible that there are additional receptors for CT and LT that are still unknown.
An affinity method has been described for CT previously which employs the GM1 oligosaccharide as the ligand (Tayot et al., (1981) Eur. J. Biochem., 113, 249-258.). However, this affinity matrix is not commercially available and the protein must be eluted from the column under extremely acidic conditions. Another method has been described based on the affinity of CT and LT for D-galactose (Uesaka et al., (1994) Microb. Path., 16, 71-76). Like the Ni-NTA agarose, this column matrix is commercially available and elution is performed under mild conditions. The method described in this patent application provides yet another affinity purification method for CT and CTB.